Among the most commonly used microscopic techniques for imaging whole cells or other materials in biology or materials science are UV-visible light microscopy or transmission electron microscopy (TEM). UV-visible light microscopy has the advantage of being able to image under ambient conditions and thus able to image dynamic processes such as cell dynamics. However, UV-visible light microscopy has limited resolution. TEM provides excellent resolution, however, in the case of biological samples, extensive preprocessing is required and the imaging must be done under vacuum. In the case of imaging cells with TEM, the cells usually must be dehydrated, embedded in plastic, and then ultra thin sections (10-100 nm) of the cells must be prepared for separate imaging owing to the limited depth of focus when using electrons.
Recently, microscopic imaging using soft x-rays has shown promise. Samples have been imaged using soft x-rays using both scanning transmission x-ray microscopy (STXM), where a sample is rastered through the source beam and the intensity of x-rays transmitted through the sample is measured point-by-point, and transmission x-ray microscopy (TXM), where full field transmission of x-rays through a sample is detected using a CCD (charge-coupled device) camera. Imaging of whole cells with soft x-rays may be accomplished by rapid freezing of fully hydrated cells. Thus, no preprocessing is required as in TEM, and high resolution approaching 20 nm can be obtained.
Owing to the need that samples in x-ray microscopy be cryogenically frozen and maintained, x-ray microscope stages require a means for continuous cooling of the sample. Previous methods have included placing a liquid nitrogen bath below the sample, thermal conduction from a liquid nitrogen bath to the sample holder, or providing a stream of liquid nitrogen cooled helium gas to the sample. These methods lack precise temperature control and may require gas stream rates that could disturb the sample during imaging. Thus, there is a need for improved cryogenic x-ray microscope stages.
Three-dimensional imaging of samples has been accomplished using light, TEM, and x-ray microscopic techniques. For example, 3D imaging using light microscopy has been conducted using confocal, two-photon confocal, through-focus deconvolution, and interferometric methods. In the case of TEM, individually imaged sections can be reconstructed to produce a 3D image. In the case of x-ray microscopy, 3D images can be constructed using computed tomography. Tomography has been accomplished with x-ray microscopy by taking a series of images (either using STXM or TXM) at different sample tilt angles. In order for the computed tomography algorithms to function properly, the images must be aligned relative to the same rotation axis. Previously, such alignment has been accomplished by either re-aligning the sample between each image or by including fiducial markers with the sample and then using a 3D marker module to align the images. However, these techniques require tedious and time-consuming manual procedures and may introduce additional error into the resulting image. Additionally, the use of fiducial markers may interfere with the sample. Accordingly, fast and automated sample alignment for tomographic x-ray microscopy is needed.